Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality

Abstract

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8 + T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8 + T cell clonotypes (CD8 EXP ). Gene expression of CD8 EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8 EXP , independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8 EXP into Jurkat 76 cells (TCR –/– ) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8 EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8 EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8 EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8 + T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution

“…A third possibility is that CD8 EXP clonotypes are cross-reactive, recognizing both recipient HLA:viral peptide ligands and simultaneously recognizing donor HLA peptides in the context of donor HLA presentation, as recently demonstrated in a mouse heart transplantation model (68). Although prior studies on kidney allograft rejection defined the intragraft CD8 EXP as alloreactive (37), it remains possible that some of the CD8 UNEXP T cell clones within our dataset are either directly alloreactive or contribute to the alloresponse by providing costimulatory 'help' to alloreactive CD8 EXP clones, as recently shown in a mouse skin transplantation model (69). The avidity, allospecificity, viral peptide cross-reactivity, and potential cooperation between CD8 EXP vs. CD8 UNEXP T cell clones is a subject of ongoing investigation utilizing human CDR3-expressing Jurkat 76 T cell lines recently described by our group (37).…”

“…Single cell RNA sequencing analysis of adult kidney allograft rejection has recently revealed that a limited number of expanded CD8 + T cell clones (CD8 EXP ) are found in the tissue and urine during ACR and that these expanded clones persist in the tissue after rejection treatment, changing their gene expression profiles in response to differing immunosuppressive regimens (37). Similar findings were recapitulated in bronchioalveolar lavage fluid from rejecting lung transplant recipients (38).…”

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell–mediated Rejection

“…Altogether, our novel findings support the hypothesis that CD8 T cells are candidate targets to treat TCMR more effectively and this postulate is also supported by our finding that the fraction of CD8 T cells is the highest among graft infiltrating T-cell subsets (Figure 9) and the recent finding from the scRNA-Seq study that CD8 T are the dominant T-cell subtype in the rejecting kidney allograft. 58 CD8 Treg have been described, 59 and CD8 targeting, therefore, should be undertaken with appropriate immune monitoring.…”

Gaining Deeper Insights Into Mechanisms of T Cell–Mediated Acute Kidney Graft Injury

“…Results of this work have raised intriguing questions about the capacity of donor-reactive T cells infiltrating kidney grafts to mediate acute rejections in patients treated with either tacrolimus, belatacept, or blocking anti-CD40 monoclonal antibody iscalimab maintenance immunosuppression. 7 The authors isolated cells from biopsies of 10 kidney grafts at the time of acute T cell-mediated rejection and several times after treatment for the rejection episode. Combined single-cell RNA sequencing and sequencing of both TcR α and β chains provided a thorough functional transcriptomic picture of the effector functions expressed by infiltrating innate and T-cell populations within the rejecting grafts.…”

“…A study from Shi et al 7 has provided new mechanistic insights to answer these important questions. Results of this work have raised intriguing questions about the capacity of donor-reactive T cells infiltrating kidney grafts to mediate acute rejections in patients treated with either tacrolimus, belatacept, or blocking anti-CD40 monoclonal antibody iscalimab maintenance immunosuppression.…”